Small-scale, high-throughput method for plant N-glycan preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis |
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Authors: | Kouki Matsuo |
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Institution: | Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan |
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Abstract: | A simple, small-scale, and high-throughput method for preparation of plant N-glycans for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) is described. This method entailed the extraction of soluble proteins, pepsin digestion, release of N-glycans by glycopeptidase A, and a three-step chromatographic purification process using cation exchange, anion exchange, and graphitized carbon. Homemade minicolumns using commercially available filter unit devices were used for N-glycan purification steps. All purification steps were designed to be easy. Using this method, N-glycans from 10-mg leaf samples of different plant species and only 2 μg of pure horseradish peroxidase were successfully purified. |
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